The present invention refers to detoxified immunogenic derivatives of Clostridium perfringens xcex2-toxin, DNA encoding such derivatives, Clostridium perfringens bacteria comprising DNA encoding such derivatives, Gram positive bacterial expression systems comprising DNA encoding such derivatives, non-Clostridium perfringens-based Gram positive bacterial expression systems expressing wild-type xcex2-toxin, vaccines for combating Clostridium perfringens based thereon, methods for the preparation of native Clostridium perfringens xcex2-toxin, methods for the preparation of detoxified immunogenic derivatives of Clostridium perfringens xcex2-toxin and to methods for the preparation of vaccines for combating Clostridium perfringens. 
Clostridium perfringens (also known as C. welchii) is a species of the large genus Clostridium. All bacteria belonging to this genus are spore-forming, anaerobic, Gram positive bacilli. The species C. perfringens can be subdivided into subspecies. Five subspecies have been described. These subspecies are generally known as xe2x80x9ctypexe2x80x9d A-E. All subspecies produce several toxins, both major and minor toxins. The four major toxins are the xcex1, xcex2, xcex5 and "igr" toxin. All C. perfringens types produce the xcex1-toxin. The xcex2-toxin is produced by C. perfringens types B and C. In addition, a range of minor toxins is produced by all C. perfringens types.
It is mainly due to the presence of one or more of these various toxins in the five C. perfringens types that all C. perfringens species are pathogenic. Type A is known to be pathogenic for both man and pigs. Type B is mainly pathogenic for lamb, sheep and goat, and causes xe2x80x9clamb dysenteriaxe2x80x9d and haemorragic enteritis. Type C is pathogenic for man, sheep, calf, lamb, pig, and bird. It is the cause of xe2x80x9cstruckxe2x80x9d, haemorragic enteritis, necrotic enteritis and enterotoxemia.
As mentioned above, both C. perfringens type B and type C are known to produce the xcex2-toxin. The xcex2-toxin is known to play the major role in the pathogenesis of necrotic enteritis in both man and animal. In man, this disease has been termed pigbel (Johnson et al.: Lancet ii: 496-500, (1987)). In animals, necrotic enteritis has been described in calves, lambs and pigs (Hauschild, A. H. W. in S. Kadis, T. C. Montie, (ed.) Microbial toxins, p. 159-188. Academic press, Inc, New York (1971) and Willis, T. A. Anaerobic bacteriology: clinical and laboratory practice, 3rd ed. Butterworths, London (1977).
The xcex2-toxin from Clostridium perfringens has been isolated in pure form (Sakurai et al., Infect. and Immun. 18: 741-745 (1977), Sakurai et al., Toxicon 25: 1301-1310 (1987)). Much is still unknown about the biophysical properties of the toxin and thus about its mode of action. Due however to the fact that it could be obtained in a purified form the toxicity of xcex2-toxin could be clearly demonstrated in animals. The lethal toxicity of purified xcex2-toxin for e.g. mice and guinea-pigs was shown by Sakurai et al. (Infect. and Immun. 21: 678-680 (1978), Sakurai et al., Toxicon 25: 1301-1310 (1987)).
Recently, the nucleic acid sequence of the Clostridium perfringens xcex2-toxin has been elucidated by Hunter et al. (Infect. and Immun. 61: 3958-3965 (1993)). The nucleic acid sequence revealed the size of the xcex2-toxin protein to be about 35 kD.
Due to the fact that the role of Clostridium perfringens xcex2-toxin from type B and C in pathogenesis is of such paramount importance, much effort has been put in the development of immunity against this toxin. Immunity against the xcex2-toxin is sufficient to protect against Clostridium perfringens type B and type C infection. The only way of inducing immunity against the xcex2-toxin is to administer the toxin to the animal to be protected. It is however obvious that the toxin must be given in a detoxified form, since otherwise administration would lead to severe illness or death of the animal.
Vaccines based on detoxified xcex2-toxin, also called xcex2-toxoid, were available already around 1960 (e.g. G.B. Pat. No. 901,433, and U.S. Pat. No. 3,288,680).
All currently available vaccines based on inactivated Clostridium perfringens xcex2-toxin have, however, several important drawbacks. In the first place, all xcex2-toxin-based vaccines comprise chemically detoxified, mainly formalin-detoxified, xcex2-toxin and it was shown through the years that it is very difficult to standardise these chemical detoxification processes. The classical chemical methods for detoxification of proteins have the disadvantage that they alter the overall structure of the protein in a fairly random manner. And as a result, during the process of chemical detoxification the immunogenic properties of the xcex2-toxin also rapidly decrease. This can be seen e.g. from FIG. 5A and FIG. 5B: in FIG. 5B it is shown that at least 1% formalin, a very commonly used inactivation-compound, is necessary to detoxify the xcex2-toxin under certain standard conditions.
From FIGS. 5A and B, it can be seen, however, that the antigenicity titre decreases dramatically with an increasing amount of formalin. A full titre cannot be obtained anyway, because even the lowest amount of formalin needed for detoxification (FIG. 5B) already gives a decrease of the titre. This implicates that there is only a very narrow band, in which both detoxification and a reasonable titre and thus immunogenicity can be obtained. Given this very narrow band, and the fact that there are at least three variables: time of detoxification, temperature and precise formalin-concentration, it is clear that it is very difficult to reproducibly produce detoxified xcex2-toxin. Another approach for the detoxification of xcex2-toxin is therefore highly wanted. No acceptable alternative has been found until now, for the following reason: the delicate balance between a sufficient level of detoxification and remaining immunogenicity implicates a close link between, on the one hand, the structure of the protein and, on the other hand, the biological properties of the protein. It could therefore not be expected to change the protein structure to detoxify the protein, without at the same time significantly impairing the immunogenic characteristics of the protein.
It is one of the merits of the present invention that it discloses for the first time a way to avoid the above-mentioned problem: using genetic manipulation techniques, specific and relatively large amino acid regions were surprisingly found that can be mutated to provide the desired non-toxic derivatives of xcex2-toxin without significantly impairing its necessary immunogenic properties.